Temporal resolution of autophosphorylation for normal and oncogenic forms of EGFR and differential effects of gefitinib.

1Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA.

Abstract

Epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases (RTK). EGFR overexpression or mutation in many different forms of cancers has highlighted its role as an important therapeutic target. Gefitinib, the first small molecule inhibitor of EGFR kinase function to be approved for the treatment of nonsmall cell lung cancer (NSCLC) by the FDA, demonstrates clinical activity primarily in patients with tumors that harbor somatic kinase domain mutations in EGFR. Here, we compare wild-type EGFR autophosphorylation kinetics to the L834R (also called L858R) EGFR form, one of the most common mutations in lung cancer patients. Using rapid chemical quench, time-resolved electrospray mass spectrometry (ESI-MS), and Western blot analyses, we examined the order of autophosphorylation in wild-type (WT) and L834R EGFR and the effect of gefitinib (Iressa) on the phosphorylation of individual tyrosines. These studies establish that there is a temporal order of autophosphorylation of key tyrosines involved in downstream signaling for WT EGFR and a loss of order for the oncogenic L834R mutant. These studies also reveal unique signature patterns of drug sensitivity for inhibition of tyrosine autophosphorylation by gefitinib: distinct for WT and oncogenic L834R mutant forms of EGFR. Fluorescence studies show that for WT EGFR the binding affinity for gefitinib is weaker for the phosphorylated protein while for the oncogenic mutant, L834R EGFR, the binding affinity of gefitinib is substantially enhanced and likely contributes to the efficacy observed clinically. This mechanistic information is important in understanding the molecular details underpinning clinical observations as well as to aid in the design of more potent and selective EGFR inhibitors.

A-B. Gefitinib effect on autophosphorylation of wild type (C) and L834R (D) EGFR. Data were fit to equation 2 as in Experimental Procedures to determine the concentration of C. Bar graph representation of the effect of gefitinib on wild type and L834R EGFR autophosphorylation. The number over the bars indicate fold difference in relative IC50 values between wild type and L834R EGFR.

Gefitinib binding to unphosphorylated and phosphorylated forms of wild type and L834R EGFR. 50 nM EGFR was titrated with increasing concentration of gefitinib and the intrinsic fluorescence signal of EGFR was quenched. The percent EGFR bound was calculated according to procedures outlined in Experimental Procedures.